Project Summary Decades of studies have suggested that pulmonary neuroendocrine cells perform multiple functions including oxygen sensing, mechanotransduction, regulation of pulmonary blood flow, regulation of airway diameter, chemosensation, and most recently the regulation of inflammation. Additionally, increased NE cell abundance has been documented in several respiratory diseases including pulmonary hypertension, neuroendocrine hyperplasia of infancy (NEHI), sudden infant death syndrome, asthma, bronchopulmonary dysplasia, congenital pneumonia, cystic fibrosis, COPD, and congenital diaphragmatic hernia. However, it remains unclear whether these NE cells are a cause of disease or whether they represent a protective regenerative response to injury. Prior research has largely been conducted on neuroendocrine bodies (NEBs) which are clustered NE cells occurring at airway branch points. Herein, we focus on the role of solitary neuroendocrine cells which may represent a distinct population of NE cells that, like NEBs, are increased in diseases associated with NE cell hyperplasia. We provide evidence that hypoxia, frequently encountered in severe lung disease, stimulates neuroendocrine differentiation as a protective response. Using lineage tracing, we will test whether hypoxia-induced NE cells are plastic and capable of differentiating into other cell types following injury. We will also assess whether NE cell hyperplasia is reversible following the restoration of normoxia using a novel airway explant system coupled to long term 2 photon imaging. We hypothesize that hypoxia-dependent neuroendocrine differentiation is mediated through the HIF signaling cascade localized to the basal stem cell compartment, and will test whether airway stem cells possess this oxygen sensing machinery which in turn is responsible for triggering the onset of NE cell differentiation. We will further examine whether HIF signaling independent of hypoxia is required for the normal maintenance of tissue resident NE cells. Using genetic cell ablation, we will assess the functional relevance of hypoxia-induced neuroendocrine differentiation in injury models. We will then test whether the abundant peptide CGRP is a protective neuropeptide that ameliorates hypoxia-induced epithelial injury by promoting progenitor cell proliferation and preventing cell death. To address NE cell heterogeneity, we have identified Tuj1 as an epithelial marker of solitary NE cells that is absent in NEBs. We show that hyperplastic NE cells in Neuroendocrine Hyperplasia of Infancy (NEHI) are positive for this marker. Lastly, we will establish a system for studying human primary airway stem cell differentiation into Tuj1+ solitary NE cells following hypoxia.